From theory to therapy: implications from an in vitro model of ramified microglia

Microglia are the principal immune cells in the central nervous system (CNS), characterized by a highly specific morphology and unusual antigenic phenotype. An increasing number of studies have focused on the role of microglia in the pathogenesis of neurodegenerative diseases. To elucidate the function of microglial cells under several neuropathological conditions, we have studied and established a cell culture model that allows us to cultivate microglial cells in their inactive, resting (ramified) phenotype. In the first part of this work, we describe the interaction of microglia cells with their epithelial (astrocytic) microenvironment. The second part reviews experiments with microglia cell cultures to elucidate underlying signalling pathways and summarizes recent advances of our knowledge in microglial molecular pathways that may ultimately lead to neurodegeneration.     

Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape

Characteristics of human malignant glioma are excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumor immune surveillance. Transforming growth factor-beta (TGF-beta), a versatile cytokine, is intimately involved in the regulation of these processes. Here, we discuss the interactions of TGF-beta with growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF), metalloproteinases (MMP-2, MMP-9) and their inhibitor, plasmin activator inhibitor-1 (PAI-1), and immune cells, like natural killer cells, T-cells and microglia. The differential effects of TGF-beta in glioma biology are outlined with emphasis on the induction of a survival advantage for glioma cells by enforced cell growth, migration, invasion, angiogenesis and immune paralysis. By virtue of its growth regulatory and immunomodulatory properties, TGF-beta promises to become a novel target for the experimental therapy of human malignant glioma.     

Ion channels in cultured microglia

Inward and, depending on activation state, outward potassium currents are the dominant ion channels in microglial cells in culture. During transition between resting and activated phases, there is also an upregulated expression of stretch/swelling-activated chloride currents. Pharmacological blockade of the specific potassium channels does not prevent the transition, whereas blockade of chloride channels does, suggesting that this current may be involved in phase changes. Interestingly, this chloride current is far less studied than the potassium currents with regard to the different microglial phases. One puzzling finding when studying microglial state is that despite changes in current densities and membrane oscillations during transition, there is no evidence of an accompanying change in membrane potential. In other cells of the immune system, membrane oscillations and alterations in membrane potential are correlated with transitions in cellular phases. This discrepancy in microglia may be a result of the fact that almost all ion channel and membrane potential studies in culture are undertaken with concomitant dialysis of cytoplasm with pipette solution. Further complicating matters is that the few studies that use microglia in situ, find fundamental differences in ion channel current patterns of "resting" microglia as well as different temporal changes to pathological events or stimuli.     

Phagocytic properties of microglia in vitro: implications for a role in multiple sclerosis and EAE.

The microglial cell, after many years of neglect, has become recognized as the sole representative cell of the immune system that resides in the normal central nervous system. While normally dormant, microglia can be activated by secretory substances or signals associated with disease or injury, and becomes a phagocytic cell, which also produces its own injurious molecules. In the activating process, its morphology is changed from a resting process-bearing cell, into a rounded amoebic form, and displays new or increased amounts of functional markers, such as receptors and Class I and Class II MHC molecules. Microglia prepared from newborn mice or rats for tissue culture are already activated, and can be used for studies of their phagocytic properties. Although they can phagocytize foreign substances, their uptake and metabolism of myelin are emphasized here, in keeping with their role in demyelinating diseases. A number of receptors have been implicated and appear to be important in the attachment to, and ingestion of, myelin particles in vitro, including the Fc, complement, macrophage scavenger, and the Galectin-3/MAC-2 receptors, although the alpha2-macroglobulin/low-density lipoprotein receptor and mannose receptors have also been suggested as participants in myelin phagocytosis. Certain cytokines and adhesion molecules also regulate the phagocytic activity of microglia. Comparative in vitro studies of phagocytosis by peritoneal macrophages and microglia have shown that the two kinds of cells respond differently to regulatory molecules, and it is concluded that they have different innate properties. The role of microglia in the demyelinative diseases experimental autoimmune encephalomyelitis and multiple sclerosis is emphasized here, and the possible means of intervention in the process leading to myelin destruction is discussed. Published 2001 Wiley-Liss, Inc.     

Review: Studies on the reproductive and developmental biology of <I>Cichlasoma dimerus</I> (Percifomes,Cichlidae)

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.     

Biochemistry and molecular biology of receptors for biogenic amines in locusts.

The biochemistry and molecular biology of biogenic amines and their metabotropic receptors in insects, with a focus on locusts, is reviewed. These compounds are known to be responsible for the control of a huge variety of different behaviours. Receptors for these amines usually belong to the class of G-protein coupled receptors (GPCR) and transmit all known functions of these compounds. The physiological significance of biogenic amine neurotransmission in insects, especially in locusts is briefly summarised. Regarding the corresponding receptors, their pharmacological features and the molecular properties are described in detail.     

技术生物工程中隆鼻整形植入体的CAD/CAM

利用计算机辅助设计与制造 (CAD/CAM) ,为隆鼻整形手术探索出一种全新硅橡胶植入体的设计与制造技术 ,解决了植入体的内部贴型准确和外部造型美观问题 ,术前的仿真设计和效果预览使患者参与自身个性化设计 ,大大增加手术成功率。     

Cell biology of the adipokinetic hormone-producing neurosecretory cells in the locust corpus cardiacum.

The adipokinetic cells are neuron-like unipolar cells, the cell bodies and cell processes of which are intermingled within the glandular part of the corpus cardiacum. In Schistocerca gregaria, they produce two adipokinetic hormones, AKH-I and -II, whereas in Locusta migratoria an additional hormone, AKH-III, is present. The three AKHs are produced by the same cells and are co-localized in secretory granules. The biosynthesis and processing of the AKH prohormones to the bioactive hormones, which has been elucidated in detail for AKH-I and -II in S. gregaria, takes less than 75 min and goes on continuously. In older locusts in particular, the adipokinetic cells contain intracisternal granules, widely dilated cisternae of the rough endoplasmic reticulum, which function as stores of prohormones of AKH-I and -II, not of AKH-III. The adipokinetic cells are subjected to regulation by a number of neural and humoral substances, neural influences coming from secretomotor cells in the lateral part of the protocerebrum. Flight activity is the only natural stimulus unequivocally shown to induce the release of AKHs, which in L. migratoria results in parallel secretion of all three AKHs. During secretory stimulation, young secretory granules containing newly synthesized hormones are preferentially released over older granules. Secretory stimulation is not accompanied by a clear increase in the levels of the AKH mRNAs and the AKH prohormones and in the rate of synthesis of the (pro-)AKHs. Apparently, a coupling between release and biosynthesis of the AKHs in the adipokinetic cells is very loose or does not even exist.     

Role of the actin cytoskeleton in insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3-kinase (PI 3-Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3-kinase. We observed recruitment PI 3-kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin-induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c-fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway.     

Low-temperature scanning electron microscopy in biology

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.     

Virtual electron microscopy in cell biology

Virtual microscopy of histological glass slides can emulate conventional light microscopy. Up till now, such a digital simulation does not exist for ultrathin electron microscopic slides. Because of the relative inaccessibility of electron microscopy, evaluation of subcellular structures by (bio)medical students is performed with the aid of photographic prints. In this article, the generation and evaluation of virtual electron microscopic slides is discussed. A T‐lymphoblastic cell was used as an example. Electron microscopic pictures were taken at two magnifications (25,000 and 50,000), processed in an analogue or digital way and stitched to reconstruct the image of the total cell. This image is viewed with a webviewer equipped with pan and zoom functions. The possibility of distinguishing the trilaminar structure of cellular membranes was the requisite. Virtual images obtained at an original magnification of 25,000, scanned at a resolution of 800 ppi could compete with pictures developed directly from negatives obtained by electron microscopy. It is possible to navigate and zoom into details in a way emulating electron microscopy. Virtual electron microscopy is innovative and offers new perspectives to interpret cytological pictures and to teach cell biology in an interactive and unique way. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

合成生物学在紫杉醇合成中的应用

合成生物学是以工程学思想为指导,对天然生物系统进行重新设计与改造,同时设计并合成新的生物元件、模块和系统的崭新学科。传统基因工程手段以及化学合成法均难以有效的实现紫杉醇的大量合成,合成生物学的发展对紫杉醇的大量合成起到了重要作用。     

Microglial interaction with beta-amyloid: implications for the pathogenesis of Alzheimer's disease.

The etiology of Alzheimer's disease (AD) involves a significant inflammatory component as evidenced by the presence of elevated levels of a diverse range of proinflammatory molecules in the AD brain. These inflammatory molecules are produced principally by activated microglia, which are found to be clustered within and adjacent to the senile plaque. Moreover, long-term treatment of patients with non-steroidal anti-inflammatory drugs has been shown to reduce risk and incidence of AD and delay disease progression. The microglia respond to beta-amyloid (Abeta) deposition in the brain through the interaction of fibrillar forms of amyloid with cell surface receptors, leading to the activation of intracellular signal transduction cascades. The activation of multiple independent signaling pathways ultimately leads to the induction of proinflammatory gene expression and production of reactive oxygen and nitrogen species. These microglial inflammatory products act in concert to produce neuronal toxicity and death. Therapeutic approaches focused on inhibition of the microglial-mediated local inflammatory response in the AD brain offer new opportunities to intervene in the disease.     

生物检测仪中数据采集模块的设计

设计生物检测仪前端的数据采集模块,采用微弱信号检测理论设计高灵敏度的电流-电压转换电路,优化硬件电路,有效降低噪声和干扰,保证数据采集的精确度.     

Optimizing sampling designs for volume measurements of components of human brain using a stereological method

Measurements of the cerebral cortical volume used to be very laborious, due to the 3-D complexity of the gyral pattern. Using stereological methods, which allow the quantification of 3-D structures from measurements on 2-D cross-sections, the difficulties have been overcome. In thirty formalin-fixed normal human brains the total volumes were measured by saline displacement. The brains were serially sliced in coronal sections and the fractional areas of the cortex, white matter, central grey structures and ventricles were determined by point-counting. Using Cavaliéri's principle the volumes of these structures were calculated. The average cortical fixed volume was 549 ml (SD + 107) corresponding to 54% of the total volume of the hemispheres. The coefficient of error of the cortical volume determinations was 2.6%. The efficiency of the design and the possibilities for optimizing the design are discussed. This point-counting method was preferred to the use of an automatic image analyser, being precise, easy to handle and not interfering with further tissue processing for histological preparation.